ORIGINAL ARTICLE
ISSN No 2230-7885 Isolation and Identification of CODEN JPBSCT NLM Title J Pharm Biomed Sci Antioxidant Flavonoids from DOI http://dx.doi.org/10.20936/jpbms/160256 the Seeds of Cardiocrinum Xue Xiaa, Guo-En Wanga, Giganteum var. Yunnanense Hoi-Yan Wub, Hai-Yan Tiana*, ABSTRACT Pang-Chui Shawb*, One new biflavonoid, 3″-hydroxyrobustaflavone (1), together with four known compounds Ren-Wang Jianga* (2-5) were isolated from the seeds of Cardiocrinum giganteum var. yunnanense. The new a Institute of Traditional Chinese Medicine structure was elucidated based on the extensive spectroscopic methods and the known and Natural Products, College of Pharmacy, compounds were identified by comparison with the literatures. In addition, all of these iso- Jinan University, Guangzhou 510632, lated compounds possessed good antioxidant capacities beyond that of L-ascorbic acid. People’s Republic of China KEYWORDS Cardiocrinum giganteum var. yunnanense, biflavonoid, flavonoid, oxygen b School of Life Sciences, The Chinese radical absorbance capacity University of Hong Kong, Shatin, Hong Kong SAR, People’s Republic of China Address reprint requests to: *Ren-Wang Jiang, College of Pharmacy, INTRODUCTION Jinan University, Huangpu Avenue West 601. Flavonoids are a group of prevalent secondary metabolites which were pro- 510632 Guangzhou, duced via the phenylpropanoid biosynthetic pathway in various plants1. People’s Republic of China E-mail: [email protected]; However, the dimers, bifavonoids are limited to exist in a few families, [email protected]; which were formed through phenol-oxidative coupling of different types [email protected] of flavonoid monomers2. It was reported that both the dimer and monomer Article citation: Xia X, Wang G, Wu HY, types of flavonoids possess a variety of pharmacological properties, such as Tian HY, Shaw PC, Jiang RW. Isolation 3 4 2 antioxidant , anti-inflammatory , anti-bacterial , etc. and identification of antioxidant flavonoids Cardiocrinum giganteum var. yunnanense Leichtlin ex Elwes, belonging to the from the seeds of Cardiocrinum giganteum family of Liliaceae, is a perennial herb of Cardiocrinum (Endl.) Lindl5. The seeds var. yunnanense. J Pharm Biomed Sci of this herb is commonly called “doulingzi” and used as the succedaneum 2016;06(06):374–377. of Aristolochia fruits in treating cough6. So far, the chemical constituents and Available at www.jpbms.info the pharmacological activities of C. giganteum have rarely been investigated7. Statement of originality of work: The During our systematic investigation on the chemical constituents of 95% manuscript has been read and approved by all ethanol extract of the seeds of Cardiocrinum giganteum var. yunnanense, one the authors, the requirements for authorship have been met, and that each author believes that the new bifavonoid, 3″-hydroxyrobustaflavone (1), as well as four known ones, manuscript represents honest and original work. 3″-hydroxyamentoflavone (2)8, quercetin (3)9, and apigenin (4)10, and kae- mpferol (5)11 were isolated. Herein, we reported the isolation, structural Source of funding: This work was supported by Guangdong key scientific project elucidation and antioxidant activities of these isolated compounds. (2013A022100029) and the Health and Medical Research Fund of Hong Kong (P.C.S). MATERIALS AND METHODS Competing interest / Conflict of interest: The author(s) have no competing interests for financial General support, publication of this research, patents and royalties through this collaborative research. Melting points were obtained on an X-5 melting point apparatus reported All authors were equally involved in discussed without correction. Optical rotations were measured on a Jasco P-1020 research work. There is no financial conflict with polarimeter in MeOH solution at room temperature. HR-ESI-TOFMS was the subject matter discussed in the manuscript. obtained on an Agilent 6210 ESI/TOF mass spectrometer. IR spectra (KBr) Disclaimer: Any views expressed in this paper were recorded on a Jasco FT/IR-480 Plus Fourier transform infrared spec- are those of the authors and do not reflect the trometer. UV spectra (MeOH) solutions were tested on a Jasco V-550 UV/ official policy or position of the Department of Defense. vis spectrophotometer. NMR spectra were recorded on a Bruker AV-300 or AV-400 spectrometer. Precoated silica gel GF254 plates (Yantai Institute of Chemical Industry, YanTai, China) were used for TLC analysis. Column chromatography was carried out on silica gel (200−400 mesh, Qingdao Marine Chemical Plant, Qingdao, China), reversed-phase C18 silica gel (Welch Materials, China), and Sephadex LH-20 (Pharmacia Biotec AB, Uppsala, Sweden). HPLC analysis was carried out on Agilent 1200 series
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Received Date: 18 April 2016 – Accepted Date: 21 May 2016 – Published Online: 5 June 2016 Isolation and identification of antioxidant flavonoids 375
2230-7885 High Performance Liquid Chromatography. Preparative chromatography eluted with a gradient of CH Cl-MeOH JPBSCT 3 HPLC was conducted on HPLC-LC100 (FIRICH). All sol- (40:0→1:1) to yield three flavones (Fr. 14A to Fr. 14J). J Pharm Biomed Sci vents used in separation and analysis were of analytical Fr. 14G (2.8 g) was separated by Sephadex LH-20 and http://dx.doi.org/10.20936/jpbms/160256 grade (Shanghai Chemical Plant, Shanghai, P. R. China) preparative HPLC eluted by CH3CN-H2O (40:60) to give and chromatographic grade (Fisher Scientific, NJ, USA), compounds 3 (17 mg), 4 (12 mg) and 5 (8 mg). 25 respectively. 3″-hydroxyrobustaflavone (1): yellow powder; [α]D -3.7 (c = 1.0, MeOH); UV (MeOH) λmax (log ε): 207 Plant material (2.94), 350 (3.15) nm; IR (KBr): νmax 3423, 1613, 1172 cm−1; 1H and 13C NMR data, see Table 1; HR-ESI-MS m/z: The seeds of Cardiocrinum giganteum var. yunnanense Leichtlin − − 553.0780 [M-H] (calcd for [C30H18O11-H] : 553.0771). ex Elwes were collected at Teng-Chong city in Yun-Nan Province of China in December, 2014 and authenticated by Professor Pang-Chui Shaw using DNA identification Antioxidant assay methods. A specimen (No. 2014092001) was depos- The antioxidant activities of the flavonoids compounds ited in the Institute of Traditional Chinese Medicine and were measured using an automated oxygen radical Natural Products, College of Pharmacy, Jinan University, absorbance capacity (ORAC) assays by the method P. R. China. described previously14. The reaction was initiated with the addition of compounds, fluorescein and 2, 2’-azobis Isolation (2-amidinopropane) dihydrochloride. The analysis was The dried and powdered seeds of Cardiocrinum giganteum performed utilizing a GENios luciferase-based micro- var. yunnanense Leichtlin ex Elwes (1 kg) were extracted plate reader (Tecan, Mannedorf, Switzerland) with exci- under ultrasonic condition (25°C, 30 min) with 95% tation/emission filter pair of 485/527 nm. The results (V/V) ethanol three times. The solution was concen- were calculated as the area under the fluorescence decay trated under reduced pressure to afford a crude extract curve using Trolox as a standard and L-ascorbic acid as a (102.3 g), which was subsequently partitioned between positive control.
EtOAc and H2O. The EtOAc part (20.5 g) was subjected to silica gel column chromatography eluted with a gradient RESULTS AND DISCUSSION of CHCl3-MeOH (80:1→1:1) to yield 20 sub-fractions (Fr. 1 to Fr. 20). Fr. 8 (3.2 g) was subjected to ODS column The dried and powdered seeds of Cardiocrinum giganteum
chromatography eluted with a gradient of MeOH-H2O var. yunnanense Leichtlin ex Elwes were extracted under (1:1→3:1) to give Fr. 8A to Fr. 8G. Fr. 8D was subjected ultrasonic condition with 95% ethanol. The EtOAc par-
to preparative HPLC eluted by CH3CN-H2O (70:30) to tition of the total extract was separated using various afford compounds 1 (15 mg) and 2 (20 mg), respec- chromatography methods, i.e. silica gel, ODS, and pre- tively. Fr. 14 (1.8 g) was subjected to silica gel column parative HPLC to afford compounds 1-5 (Fig. 1).
1 13 Table 1 H and C NMR data of 1 (δ, in DMSO-d6 , J in Hz). No. 1H 13C No. 1H 13C 2 167.8 2″ 145.8 3 6.79 s 102.1 3″ 135.1 4 181.5 4″ 175.5 5 164.3 5″ 161.3 6 6.16 d (1.8) 98.7 6″ 105.3 7 165.1 7″ 163.9 8 6.37 d (1.8) 93.8 8″ 6.15 s 100.1 9 157.2 9″ 153.6 10 103.5 10″ 101.6 1′ 118.6 1″′ 122.0 2′ 8.18 d (2.4) 131.2 2″′ 6″′ 7.80 d (8.8) 129.1 3′ 122.3 3″′ 5″′ 6.58 d (8.8) 114.9 4′ 159.6 4″′ 158.6 5′ 6.96 d (8.7) 118.1 5″-OH 12.66 6′ 7.91 dd (8.7, 2.4) 126.6 5-OH 13.06
J Pharm Biomed Sci | Vol. 06 No. 06 | 374–377 376 Ren-Wang Jiang
Fig. 1 Chemical Structures of 1-5.
Fig. 4 Representative curves of fluorescence decay induced by AAPH in the presence of five compounds (4.0 μM for compounds 1-4 and 1.0 μM for compound 5) and Trolox (20 μM).
hydroxyl and carbonyl groups. After a detailed analysis of the 1D and 2D spectra of 1, the existence of one flavone and one flavonol units was confirmed (rings A-C and D-F)12. The 1H NMR spectrum of 1 showed proton sig-
nals at δH 13.07 (1H, s, 5-OH), 6.16 (1H, d, J = 1.8 Hz, Fig. 2 Key HMBC correlations of 1. H-6), and 6.37 (1H, d, J = 1.8 Hz, H-8) in a 1,3,4,5 tet-
ra-substituted ring A, δH 8.18 (1H, d, J = 2.4 Hz, H-2′), 7.91 (1H, dd, J = 8.7, 2.4 Hz, H-6′) and 6.96 (1H, d, J
= 8.7 Hz, H-5′) in a 1,3, 4 tri-substituted ring B, and δH 6.79 (s) in ring C. These above evidences supported the existence of a flavone part A in 1 (Fig. 2). Accordingly, the
proton signals at δH 12.66 (1H, s, 5″-OH) and 6.15 (1H, s, H-8″) for ring D, δH 7.80 (2H, d, J = 8.8 Hz, H-2′″, H-6′″) and 6.58 (2H, d, J = 8.8 Hz, H-3′″, H-5′″) for a para-substituted ring E were in accordance with a favonol part B in 1 (Fig. 2). The 13C NMR spectrum showed thirty carbon signals, including nineteen aromatic quaternary carbons, and eleven aromatic methines, among which,
carbon signals at δC 181.5 (C-4″) and 175.5 (C-4) were two typical favonoid keto-carbonyl groups, confirming that 1 was bifavonoid type natural product containing two C6-C3-C6 units. The 1H and 13C NMR signals of 1 were all assigned unambiguously based on the 1H-1H COSY, HSQC and Fig. 3 ORAC index of the isolated compounds. The values were HMBC data (Table 1). In the HMBC spectrum, HMBC calculated as the net area under the fluorescence decay curve and correlations between H-2’ and H-6”, and between H-8’’ expressed as 20 μM Trolox equivalents. Data are expressed as and H-4’ indicated that the favone unit was connected to means ± SD from three samples in each compound. VitC rep- the favonol via a C3’-C6’’ carbon-carbon bond. Thus, the resents for L-ascorbic acid. structure of 1 was established to be 3″-hydroxyrobusta- flavone, which was a robustaflavone type bifavonoid, Compound 1 was obtained as a light yellow powder. consisting of apigenin (4) and kaempferol (5). The molecular formula of 1 was established to be The antioxidant activities of these isolated com-
C30H18O11 based on a quansi-molecular ion peak at m/z pounds were evaluated using oxygen radical absorbance 553.0870 in its HR-ESI-TOFMS, with twenty-two degrees capacity antioxidant assay (ORAC) (Figs. 3 and 4). The of unsaturation. The UV spectrum showed characteristic results showed that either the monomers or the dimers absorptions of flavonoids at 269 and 350 nm. The IR exhibited much higher antioxidant activities than the bands at 3432 and 1613 cm−1 indicated the existence of positive control L-ascorbic acid. At the concentrations
J Pharm Biomed Sci | Vol. 06 No. 06 | 374–377 Isolation and identification of antioxidant flavonoids 377 of 4.0 μM, the dimers, 1 and 2 were 1.5 times more phytochemical prevents dextran sulphate sodium-induced active than L-ascorbic acid at 40.0 μM. By contrast, the colitis in rats. Basic Clin Pharmacol Toxicol. 2013;113(1):49–55. monomers, 3 and 4 at the same concentration of 4.0 μM 5. Nishikawa T, Okazaki K, Uchino T, Arakawa K, Nagamine T. A showed 2.5 times of activities compared with L-ascorbic molecular phylogeny of Lilium in the internal transcribed spacer acid (40.0 μM). Compound 5, which was the most region of nuclear ribosomal DNA. J Mol Evol. 1999;49(2):238–249. potent one, at a lower concentration of 1.0 μM are 4 6. Li M, Ling KH, Lam H, Shaw PC, Cheng L, Techen N, Khan LA, Chang YS, But PP. Cardiocrinum seeds as a replacement for times more active than L-ascorbic acid (40.0 μM). These Aristolochia fruits in treating cough. J Ethnopharmacol. 2010; above results indicated that although all compounds are 130(2):429–432. shown to be potential antioxidant agents, the monomers 7. Wang YZ, Li SB, Guo HC, Fan HC, Sha BC, Chen FS, Wang JH. are more favorable for the antioxidant activities. [Determination of trace elements in Cardiocrinum giganteum by FAAS] Guang Pu Xue Yu Guang Pu Fen Xi 2007;27 (9):1854–1857. CONCLUSION 8. Chen YG, Yu LL, Huang R, Liu JC, LV YP, Zhao Y. 3”- Hydroxyamentoflavone and its 7-O-methyl ether, two new Two flavonoid dimers (1-2) and three monomers (3-5) biflavonoids from Aristolochia contorta. Arch Pharm Res. 2005; were identified from the seeds Cardiocrinum giganteum, 28(11):1233–1235. which enhanced the understanding of the chemical con- 9. Torgils F, Atle T. Flavonoids from red onion (Allium Cepa). notation of Cardiocrinum giganteum. As a rich source of fla- Phytochemistry. 1998;47(2):281–285. vonoids (polyphenols) with potent antioxidant activities, 10. Miyazawa M, Hisama M. Antimutagenic activity of flavo- Cardiocrinum giganteum might be developed to anti-oxidative noids from Chrysanthemum morifolium. Biosci Biotech Bioch. 2003;67(10):2091–2099. products for anti-aging, relieving oxidative stress, etc13. 11. Ding HY, Lin HC, Teng CM, Wu YC. Phytochemical and pharma- cological studies on Chinese Paeonia species. J Chin Chem Soc- REFERENCES Taip. 2000;47(2):381–388. 12. Ito T, Yokota R, Watarai T, Mori K, Oyama M, Nagasawa H, 1. Chen WH, Wang R, Shi YP. Flavonoids in the poisonous plant Matsuda H, Iinuma M. Isolation of Six Isoprenylated Biflavonoids Oxytropis falcata. J Nat Prod. 2010;73(8):1398–1403. from the Leaves of Garcinia subelliptica. Chem Pharm Bull. 2. Messi BB, Ndjoko-Ioset K, Hertlein-Amslinger B, Lannang AM, 2013;61(5):551–558. Nkengfack AE, Wolfender JL, Hostettmann K, Bringmann G. 13. Farombi EO, Owoeye O. Antioxidative and chemopreventive Preussianone a new flavanone- chromone biflavonoid from properties of Vernonia amygdalina and Garcinia biflavonoid. Int Garcinia preussii Engl. Molecules 2012;17(5):6114–6125. J Environ Res Pub Health. 2011;8(6):2533–2555. 3. Miceli N, Trovato A, Dugo P, Cacciola F, Donato P, Marino A, 14. Farombi EO, Adedara IA, Akinrinde SA, Ojo OO, Eboh AS. Bellinghieri V, La Barbera TM, Guvenc A, Taviano MF. Comparative Protective effects of kolaviron and quercetin on cadmium- analysis of flavonoid profile, antioxidant and antimicrobial activ- induced testicular damage and endocrine pathology in rats. ity of the berries of Juniperus communis L. var. communis and Andrologia. 2012;44(4):273–284. Juniperus communis L. var. saxatilis Pall. from Turkey. J Agric 15. Wang GE, Li YF, Wu YP, Tsoi B, Zhang SJ, Cao LF, Kurihara H, He Food Chem. 2009;57(15):6570–6577. RR. Phloridzin improves lipoprotein lipase activity in stress- 4. Farombi, EO, Adedara IA, Ajayi BO, Ayepola OR, Egbeme loaded mice via AMPK phosphorylation. Int J Food Sci Nut. EE, Kolaviron. A natural antioxidant and anti-inflammatory 2014;65(7):874–880.
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